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Abstract The transport of live fish is a routine practice in aquaculture and constitutes a considerable source of stress to the animals. The addition of anesthetic to the water used for fish transport can prevent or mitigate the deleterious effects of transport stress. This study investigated the effects of the addition of eugenol (EUG) (1.5 or 3.0 µL L–1) and essential oil of Lippia alba (EOL) (10 or 20 µL L–1) on metabolic parameters (glycogen, lactate and total protein levels) in liver and muscle, acetylcholinesterase activity (AChE) in muscle and brain, and the levels of protein carbonyl (PC), thiobarbituric acid reactive substances (TBARS) and nonprotein thiol groups (NPSH) and activity of glutathione-S-transferase in the liver of silver catfish (Rhamdia quelen; Quoy and Gaimard, 1824) transported for four hours in plastic bags (loading density of 169.2 g L–1). The addition of various concentrations of EUG (1.5 or 3.0 µL L–1) and EOL (10 or 20 µL L–1) to the transport water is advisable for the transportation of silver catfish, since both concentrations of these substances increased the levels of NPSH antioxidant and decreased the TBARS levels in the liver. In addition, the lower liver levels of glycogen and lactate in these groups and lower AChE activity in the brain (EOL 10 or 20 µL L–1) compared to the control group indicate that the energetic metabolism and neurotransmission were lower after administration of anesthetics, contributing to the maintenance of homeostasis and sedation status.

RESUMO:The effects of anesthesia with the essential oil of Ocimum gratissimum (EOO) in parameters of stress after handling were investigated in silver catfish (Rhamdia quelen). EOO was obtained from the aerial parts by hydrodistillation. Juveniles were anesthetized with 70 or 300 mg L-1 EOO and submitted to air exposure for 1 minute. The fishes were sampled immediately or transferred to anesthetic-free aquaria until sampling. In the first experiment, juveniles had their blood collected at 0, 1, 4, and 8 h after handling to assay plasma cortisol and blood glucose levels. The unanesthetized animals were restrained manually for blood collection. In the second experiment, water samples of the recovery aquaria were collected to evaluate net ion fluxes at 0 - 4 h and 4 - 8 h. Water and ethanol controls were also performed under the same conditions. The results showed that the cortisol levels did not differ among the treatments. Hyperglycemia was verified in fish exposed to 70 and 300 mg L-1 EOO at 1 h and 4 h after handling. After 8 h, cortisol and glucose concentrations were lower or similar than those from immediately after handling for all treatments. EOO anesthesia prevented Na+ efflux observed in the control groups in both flux periods. There were net Cl- and K+ effluxes at 0 - 4 h and influxes at 4 - 8 h after handling in most treatments, and these fluxes did not differ among the treatments. The results suggest that EOO did not impair stress recovery and did not act as an additional handling stressor in silver catfish.

This study evaluated the anesthetic potential of thymol and carvacrol, and their influence on acetylcholinesterase (AChE) activity in the muscle and brain of silver catfish (Rhamdia quelen). The AChE activity of S-(+)-linalool was also evaluated. We subsequently assessed the effects of thymol and S-(+)-linalool on the GABAergic system. Fish were exposed to thymol and carvacrol (25, 50, 75, and 100 mg/L) to evaluate time for anesthesia and recovery. Both compounds induced sedation at 25 mg/L and anesthesia with 50–100 mg/L. However, fish exposed to carvacrol presented strong muscle contractions and mortality. AChE activity was increased in the brain of fish at 50 mg/L carvacrol and 100 mg/L thymol, and decreased in the muscle at 100 mg/L carvacrol. S-(+)-linalool did not alter AChE activity. Anesthesia with thymol was reversed by exposure to picrotoxin (GABAA antagonist), similar to the positive control propofol, but was not reversed by flumazenil (antagonist of benzodiazepine binding site), as observed for the positive control diazepam. Picrotoxin did not reverse the effect of S-(+)-linalool. Thymol exposure at 50 mg/L is more suitable than carvacrol for anesthesia in silver catfish, because this concentration did not cause any mortality or interference with AChE activity. Thymol interacted with GABAA receptors, but not with the GABAA/benzodiazepine site. In contrast, S-(+)-linalool did not act in GABAA receptors in silver catfish.

This study evaluated the sedative and anesthetic effects of the essential oils (EO) of Hyptis mutabilis (Rich.) Briq. and their isolated components on silver catfish (Rhamdia quelen). Quantitative chemical differences between the EOs obtained from leaves and inflorescences were verified, and a new chemotype rich in globulol was described. Although there were no significant differences in the time of induction for sedation and anesthesia between the EOs, only the leaf EO at 344 mg/L anesthetized all fish without side effects. Fractionation of the leaf EO was carried out by column chromatography. The isolated compounds [(+)-1-terpinen-4-ol and (-)-globulol] showed different activity from that detected for the leaf EO in proportional concentrations and similar sedation to a eugenol control at 10 mg/L. However, fish exposed to 1-terpinen-4-ol (3 and 10 mg/L) did not remain sedated for 30 min. Anesthesia was obtained with 83-190 mg/L globulol, but animals showed loss of mucus during induction and mortality at these concentrations. Synergism of the depressor effects was detected with the association of globulol and benzodiazepine (BDZ), compared with either drug alone. Fish exposed to BDZ or globulol+BDZ association showed faster recovery from anesthesia in water containing flumazenil, but the same did not occur with globulol. In conclusion, the use of globulol in aquaculture procedures should be considered only at sedative concentrations of 10 and 20 mg/L, and its mechanism of action seems not to involve the GABAA-BDZ system.

(+)-Dehydrofukinone (DHF) is a major component of the essential oil of Nectandra grandiflora (Lauraceae), and exerts a depressant effect on the central nervous system of fish. However, the neuronal mechanism underlying DHF action remains unknown. This study aimed to investigate the action of DHF on GABAA receptors using a silver catfish (Rhamdia quelen) model. Additionally, we investigated the effect of DHF exposure on stress-induced cortisol modulation. Chemical identification was performed using gas chromatography-mass spectrometry and purity was evaluated using gas chromatography with a flame ionization detector. To an aquarium, we applied between 2.5 and 50 mg/L DHF diluted in ethanol, in combination with 42.7 mg/L diazepam. DHF within the range of 10-20 mg/L acted collaboratively in combination with diazepam, but the sedative action of DHF was reversed by 3 mg/L flumazenil. Additionally, fish exposed for 24 h to 2.5-20 mg/L DHF showed no side effects and there was sustained sedation during the first 12 h of drug exposure with 10-20 mg/L DHF. DHF pretreatment did not increase plasma cortisol levels in fish subjected to a stress protocol. Moreover, the stress-induced cortisol peak was absent following pretreatment with 20 mg/L DHF. DHF proved to be a relatively safe sedative or anesthetic, which interacts with GABAergic and cortisol pathways in fish.

The objective of this study was to identify the possible involvement of the GABAergic system in the anesthetic effect of Lippia alba essential oil (EO). We propose a new animal model using silver catfish (Rhamdia quelen) exposed to an anesthetic bath to study the mechanism of action of EO. To observe the induction and potentiation of the anesthetic effect of EO, juvenile silver catfish (9.30 ± 1.85 g; 10.15 ± 0.95 cm; N = 6) were exposed to various concentrations of L. alba EO in the presence or absence of diazepam [an agonist of high-affinity binding sites for benzodiazepinic (BDZ) sites coupled to the GABA A receptor complex]. In another experiment, fish (N = 6) were initially anesthetized with the EO and then transferred to an anesthetic-free aquarium containing flumazenil (a selective antagonist of binding sites for BDZ coupled to the GABA A receptor complex) or water to assess recovery time from the anesthesia. In this case, flumazenil was used to observe the involvement of the GABA-BDZ receptor in the EO mechanism of action. The results showed that diazepam potentiates the anesthetic effect of EO at all concentrations tested. Fish exposed to diazepam and EO showed faster recovery from anesthesia when flumazenil was added to the recovery bath (12.0 ± 0.3 and 7.2 ± 0.7, respectively) than those exposed to water (9.2 ± 0.2 and 3.5 ± 0.3, respectively). In conclusion, the results demonstrated the involvement of the GABAergic system in the anesthetic effect of L. alba EO on silver catfish.

(+)-Dehydrofukinone (DHF) is a major component of the essential oil of Nectandra grandiflora (Lauraceae), and exerts a depressant effect on the central nervous system of fish. However, the neuronal mechanism underlying DHF action remains unknown. This study aimed to investigate the action of DHF on GABAA receptors using a silver catfish (Rhamdia quelen) model. Additionally, we investigated the effect of DHF exposure on stress-induced cortisol modulation. Chemical identification was performed using gas chromatography-mass spectrometry and purity was evaluated using gas chromatography with a flame ionization detector. To an aquarium, we applied between 2.5 and 50 mg/L DHF diluted in ethanol, in combination with 42.7 mg/L diazepam. DHF within the range of 10-20 mg/L acted collaboratively in combination with diazepam, but the sedative action of DHF was reversed by 3 mg/L flumazenil. Additionally, fish exposed for 24 h to 2.5-20 mg/L DHF showed no side effects and there was sustained sedation during the first 12 h of drug exposure with 10-20 mg/L DHF. DHF pretreatment did not increase plasma cortisol levels in fish subjected to a stress protocol. Moreover, the stress-induced cortisol peak was absent following pretreatment with 20 mg/L DHF. DHF proved to be a relatively safe sedative or anesthetic, which interacts with GABAergic and cortisol pathways in fish.

In view of the importance of the intestine in the osmoregulation of freshwater fishes, we determined the effects of oxytocin, urotensin II (UII), and aldosterone added to the serosal side of the isolated posterior intestine of the freshwater-adapted teleost Anguilla anguilla on electrophysiological parameters. Oxytocin decreased the short-circuit current (SCC) and transepithelial potential difference (TPD) at concentrations of 1 and 10 mU/ml (to 50% and 42% of control values, respectively), but did not alter these parameters at a concentration of 0.1 mU/ml. UII reduced SCC and TPD at concentrations of 10 nM, 50 nM and 100 nM (to 85% of control values), but increased these parameters at the concentration of 500 nM (to 115% of control values). Aldosterone did not alter SCC or TPD at the concentrations tested (10 nM and 100 nM). Oxytocin may open Na+ channels in the apical membrane, allowing the flow of Na+ to the serosa, reducing SCC and TPD. Should this hypothesis be correct, oxytocin would be important for freshwater adaptation, since it would increase Na+ absorption. The reduction of SCC and TPD in the posterior intestine of A. anguilla induced by UII is evidence that this neurohormone is also important for freshwater adaptation in teleosts. Aldosterone did not show this effect probably due to the lack of receptors in this organ

The aim of this study was to investigate the effect on an increase of temperature on the net ion fluxes on Metynnis hypsauchen, a teleost species from the Rio Negro. Fish were collected in the Anavilhanas archipelago, Rio Negro, Amazon. After 24 h adaptation fish were placed in individual chambers served with a steady flow of recirculated water. Na+ and Cl- fluxes were determined at 26 and 33ºC. After 18 h in the chambers, fish presented an influx of Na+ and Cl-, and the temperature raise to 33ºC led to an efflux of both ions, which remained even after 6 h in this temperature. Six hours were not enough to promote a significant reduction of net ion effluxes, but certainly the fluxes would be in net balance after a longer period of time, since this species can be exposed to this temperature in its natural environment.

The aim of this study was to describe the effect of hypoxia on whole body ion fluxes and hematological parameters in two Amazonian teleosts: Serrasalmus eigenmanni and Metynnis hypsauchen. The increase of Na+ and Cl- effluxes on M. hypsauchen exposed to hypoxia may be related to an increase of gill ventilation and effective respiratory surface area, to avoid a reduction in the oxygen uptake, and/or with the decrease of pHe, that could inhibit Na+ and Cl- transporters and, therefore, reduce influx of these ions. Effluxes of Na+ and Cl- were lower in hypoxia than in normoxia for S. eigenmanni, possibly because in hypoxia this species would reduce gill ventilation and oxygen uptake, which would lead to a decrease of gill ion efflux and, consequently, reducing ion loss. The increase on hematocrit (Ht) during hypoxia in M. hypsauchen probably was caused by an increase of the red blood cell volume (MCV). For S. eigenmanni the increase on glucose possibly results from the usage of glucose reserve mobilization. Metynnis hypsauchen showed to be more sensitive to hypoxia than Serrasalmus eigenmanni, since the first presented more significant alterations on these osmoregulatory and hematological parameters. Nevertheless, the alterations observed for both species are strategies adopted by fishes to preserve oxygen supply to metabolizing tissues during exposure to hypoxia.

This study aimed to evaluate the effectiveness of essential oils (EO) of Pilocarpus pennatifolius (PPOL) and Cordia verbenacea (CVOL) as sedatives and/or anesthetics in juvenile Nile tilapia (Oreochromis niloticus). The main constituents of PPOL were 2-undecanone (57.2%) and 2-Tridecanone (28.3%), and of CVOL α-pinene (34.8%), which were determined by gas chromatography. PPOL led juveniles to sedation at concentrations of 2 (703.8s), 10 (407.4s), 20 (225.5s), 50 (190.2s), and 80 (163.5s) mg L-1. CVOL also induced sedation at these concentrations, with times of 504.9, 294.2, 142.8, 113.8, and 135.6 s, respectively. Both EOs at 200 mg L-1 induced deep anesthesia within 507.5 (PPOL) and 1267.7 s (CVOL). Exposure to PPOL at 2, 20, and 80 mg L-1 for 48 h resulted in fish sedation, while CVOL lead fish to sedation at the two lowest concentrations and anesthesia at the highest one. Possible stress responses were evaluated during anesthetic recovery and long exposure, and PPOL did not cause behavioral or glucose levels changes in all evaluations. However, CVOL increased glucose levels at the highest concentrations studied (80 and 200 mg L-1), indicating that this oil may have caused stress in fish.

This study aimed to evaluate the effects of Protium heptaphyllum fruit essential oil (PHEO) on the physiology of silver catfish (Rhamdia quelen) during anesthesia and recovery, through studying echocardiograms, oxidative status, and metabolic parameters. Three experiments were performed: (1) 50 silver catfish juveniles were submitted to anesthesia and recovery tests with 300, 400, 500, 600, and 700 mg L -1 of PHEO. (2) Echocardiogram analysis was performed in anesthetized and non-anesthetized fish. (3) Biochemical parameters were evaluated at 0, 30, 60, and 120 min of recovery after being anesthetized for 3 min with 600 mg L -1 PHEO. Times to sedation and deep anesthesia were reduced with PHEO increasing concentrations. The echocardiogram showed a higher cardiac rate in anesthetized fish. Plasma glucose levels increased in control fish through recovery time, but anesthetized fish showed lower levels than controls at 120 min of recovery. Metabolic parameters such as plasma and hepatic glucose did not show changes considering the recovery time of up to 120 min. Hepatic glycogen, lactate, and triglycerides reduced their levels over recovery times. Fish anesthetized enhanced superoxide dismutase activity and thiobarbituric acid reactive substances levels but decreased reduced glutathione (GSH) levels at 30 min compared to controls. After 60 min, GSH values were significantly higher in anesthetized fish than in controls. These results suggest that PHEO at 600 mg L -1 is an effective anesthetic for the rapid handling of silver catfish, providing stable metabolic parameters and enhanced antioxidant responses during recovery. Echocardiogram analysis confirms the anesthetic effect, supporting PHEO as a viable and efficient option for fish anesthesia in aquaculture. The use of PHEO in aquaculture can enhance fish welfare by reducing stress during handling and transportation, potentially leading to improved growth, health, and survival rates., Competing Interests: Declarations Ethical approval This study was approved by the Ethic Committee on Animal Use of the Federal University of Santa Maria-Brazil (CEUA/UFSM) by number 1668061020. Consent for publication All authors consent to manuscript publication. Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The aim of this study is to analyze the waterborne metal levels in four lakes (one endorheic and three exorheic) of Harmony Point, Nelson Island, Antarctica. Water samples were analyzed by using a quadrupole type inductively coupled plasma mass spectrometer and an inductively coupled plasma optical emission spectrometer. The levels of As, Cu, Mn, Mo, and V were significantly lower and those of Cr, Mg, Na, and Sr were significantly higher in the endorheic lake than in the other lakes. Most water samples presented levels of Ag, Be, Cd, Pb, Se, Tl, and U below the limit of quantification, while for Ba, Co, and Ni around half of the samples were below this limit. The waterborne metal levels were not significantly different between the exorheic lakes. Waterborne metal levels in the freshwater lakes from Harmony Point did not show any clear relationship with their levels in the soil of the region or with bird guano, and overall, their levels indicate an environment without anthropogenic influence. Apparently, the Na levels are influenced by salt spray from the ocean, as they are related to the distance of the lakes from the ocean.

Blood analysis is an important tool for monitoring the health status of fish, but the time between collection and analysis can affect the outcome of the analysis. This study sought to determine the maximum time refrigerated blood and frozen plasma samples of the tambaqui, Colossoma macropomum, can be stored without affecting analysis. Samples from 12 fish were obtained, stored under refrigeration at 4 °C and evaluated after 0, 24, 48, 72, and 96 h, while samples from 14 fish were centrifuged, and the resulting plasma was frozen at -20 °C and then evaluated after 0, 8, 12, 16 and 20 weeks. The parameters analyzed were hematocrit (Ht), hemoglobin content (Hb), total erythrocytes (RBC), total (WBC) and differential leukocytes, total thrombocytes (TC), glucose content (Glc), total protein (TP), triglyceride content (TG), total cholesterol (CoT), and the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). For refrigerated whole blood samples, mean corpuscular hemoglobin content (MCHC) showed a transient decline in 24 h, and there were decreases in WBC, TC, Glc and TG that persisted until the 72 h sample point (for Glc and TG) or persisted until the 96 h sample point (for WBC and TC). A decrease in RBC was noted from 48 h on, while ALT was significantly higher in the 96 h sample. Significant decreases in lymphocytes, monocytes, neutrophils and eosinophils were noted from 48 h of storage on, while a significant decline in basophil counts were noted over the last two sampled timepoints. The coefficient of variation was greatest at the 96 h timepoint, indicating increased variability in measured parameters after 4 d of refrigeration. Plasma samples frozen at -20 °C showed a significant variation in ALT after 8 weeks, and increases in TP and TG after 20 weeks. Therefore, it is recommended that refrigerated tambaqui whole blood samples be analyzed within 24 h and frozen tambaqui plasma samples analyzed within 8 weeks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study evaluated the use of essential oil of Ocimum gratissimum (EOOG) for anesthesia and in transport of Colossoma macropomum. Experiment 1, Test 1, anesthesia induction and recovery times were determined using different EOOG concentrations (0, 20, 50, 100, 200, 300 mg L -1 ), with two size classes: Juveniles I (0.86 g) and Juveniles II (11.46 g) (independent tests in a completely randomized design). Based on the results of Test 1, in Test 2 Juveniles II were exposed to EOOG concentrations: 0, 20, 100 mg L -1 . Tissue samples were collected immediately after induction and 1 h post-recovery, to assess oxidative status variables. Experiment 2, Juveniles I (0.91 g) and Juveniles II (14.76 g) were submitted to transport in water with different concentrations of EOOG (0, 5, 10 mg L -1 ) (independent tests in a completely randomized design). The effects on oxidative status variables were evaluated. Concentrations between 50 and 200 mg L -1 EOOG can be indicated for Juveniles I, while concentrations between 50 and 100 mg L -1 EOOG for Juveniles II. The concentration of 100 mg L -1 EOOG was able to prevent oxidative damage in the liver. In Experiment 2, the concentrations of 5 and 10 mg L -1 EOOG added to the transport water caused sedation for both studied size classes of juveniles and did not cause oscillations in water quality variables nor any mortality. The concentration of 10 mg L -1 EOOG improved the oxidative status. It can be concluded that EOOG can be used for anesthesia and transport of C. macropomum., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Climatic events are affecting the Amazon basin and according to projections it is predicted the intensification of climate changes through increases in temperature and carbon dioxide (CO 2 ). Recent evidence has revealed that exposure to an extreme climate scenario elicits oxidative damage in some fish species, impairing their metabolism and physiology, contributing to their susceptibility. Thus, the comprehension of physiological alterations in Arapaima gigas (pirarucu) to the climatic changes forecasted for the next 100 years is important to evaluate its capability to deal with oxidative stress. The objective of this work was to determine whether antioxidant defense system is able to prevent muscle oxidative damage of pirarucu exposed 96 h to extreme climate scenario, as well as the effects of this exposition on muscle fatty acid levels. Lipid peroxidation and reactive oxygen species significantly increase in the muscle of pirarucus exposed to an extreme climate scenario compared to control, while muscle superoxide dismutase, catalase, and glutathione peroxidase were significantly lower. Total amount of saturated fatty acids (SFAs) was significantly higher in pirarucu exposed to an extreme climate scenario compared to control, while total content of monounsaturated (MUFAs) and polyunsaturated fatty acids (PUFAs) was significantly lower. Exposure to an extreme climate scenario causes muscular oxidative stress and that the antioxidant systems are inefficient to avoid oxidative damage. In addition, the increase of total SFAs and the decrease of MUFAs and PUFAs probably intend to maintain membrane fluidity while facing high temperature and CO 2 levels., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The growing increase in the fish farming sector has favored the establishment of bacterial outbreaks caused by Aeromonas hydrophila in several species. The hexane extract of Hesperozygis ringens (HEHR) (Lamiaceae) leaves increased the survival rate of silver catfish (Rhamdia quelen) experimentally infected by A. hydrophila. However, it is noteworthy that no reports have been found on the possible mechanisms of action of this extract in infected fish. This study aimed to evaluate the effect of the HEHR, administered through single immersion bath, on lipid peroxidation and antioxidant defenses in muscle and liver tissue of silver catfish challenged with A. hydrophila. The results showed that the oxidative status of silver catfish was altered, although oxidative stress was not triggered during the experiment. HEHR at 30 mg/L (HEHR30) was not characterized as a pro-oxidant agent in the presence of infection, unlike florfenicol and HEHR at 15 mg/L treatments in some cases. In short, HEHR30 provided an important increase in hepatic catalase activity, characterizing one of the possible mechanisms involved in the greater survival of fish experimentally infected by A. hydrophila. Additionally, HEHR30 did not induce lipid peroxidation, nor reduced antioxidant defenses of silver catfish infected or not by A. hydrophila.

This study aimed to identify the regions of the body surface of silver catfish ( Rhamdia quelen ) with a higher population of mucous cells in the skin. Additionally, the effects of stressful conditions, such as different stocking densities and food deprivation, on the proliferative response of mucous cells in the skin and gill epithelium and their impact on cutaneous mucous lysozyme activity were investigated. Silver catfish were divided into four experimental groups: high stocking density (32 kg/m 3 ) and fed (HSD-F), high stocking density and fasted (HSD-FS), low stocking density (2.5 kg/m 3 ) and fed (LSD-F), and low stocking density and fasted (LSD-FS). Fish in the fed groups received commercial feed twice a day, amounting to 1% of the tank biomass. After a 14-day experimental period, the fish were anesthetized and euthanized. Samples of cutaneous mucous and skin fragments from seven different points and the second left branchial arch were collected. Histological slides of the skin and gills were stained with PAS + Alcian Blue at pH 2.5, and the epidermal mucous lysozyme activity was assessed using the turbidimetric method. The ventral point in front of the ventral fin was found to be the optimal location for collecting cutaneous epithelia due to its higher density of mucous cells. The population of mucous cells in both the skin and gills varied based on the collection point and treatment applied. The highest lysozyme activity in the epidermal mucous was observed in fish from the HSD-F group. Overall, these findings suggest that stocking density and food deprivation create stressful conditions for silver catfish, which modulate their mucosal response to each situation.

R-(+)-limonene is a monoterpene from plants of the genus Citrus with diverse biological properties. This research evaluated the effects of dietary supplementation with R-(+)-limonene on growth, metabolic parameters in plasma and liver, and the antioxidant and stress responses in silver catfish, Rhamdia quelen , challenged or not with Aeromonas hydrophila . Fish were fed for 67 days with different doses of R-(+)-limonene in the diet (control 0.0, L0.5, L1.0, and L2.0 mL/kg of diet). On the 60th day, a challenge with A. hydrophila was performed. R-(+)-limonene in the diet potentiated the productive performance of the fish. The metabolic and antioxidant responses indicate that R-(+)-limonene did not harm the health of the animals and made them more resistant to the bacterial challenge. Histological findings showed the hepatoprotective effect of dietary R-(+)-limonene against A. hydrophila . Igf1 mRNA levels were upregulated in the liver of fish fed with an L2.0 diet but downregulated with bacterial challenge. The expression levels of crh mRNA were higher in the brains of fish fed with the L2.0 diet. However, the L2.0 diet downregulated crh and hspa12a mRNA expression in the brains of infected fish. In conclusion, the results indicated that R-(+)-limonene can be considered a good dietary supplement for silver catfish.

The aim of this study was to determine the biological effects of dietary supplementation with 0.05% and 0.1% cinnamon essential oil extracted from Cinnamomum cassia on silver catfish ( Rhamdia quelen ). The final body weight, weight gain, and specific growth rate were significantly higher in fish supplemented with 0.05% cinnamon essential oil than in the control(untreated) group. Muscle reactive oxygen species and lipid peroxidation levels were significantly lower in fish supplemented with 0.05% cinnamon essential oil but higher at the 0.1% concentration. Muscle antioxidant capacity against peroxyl radicals (ACAP) and superoxide dismutase activity were significantly higher in fish supplemented with 0.05% cinnamon essential oil, while ACAP levels were lower in fish supplemented with 0.1%. The total saturated fatty acid content was significantly higher in the muscle of supplemented fish than in controls, while the total monounsaturated fatty acid content was significantly higher only in fish fed 0.1% cinnamon essential oil. Finally, the total content of polyunsaturated fatty acids was significantly lower in fish fed 0.1% essential oil. Thus, data demonstrated that 0.05% C. cassia essential oil improves fish health by improving performance and muscle oxidant/antioxidant status. Higher doses of cinnamon essential oil produced oxidative stress in muscle, suggesting toxicity at the 0.1% level. Although this cinnamon essential oil diet exerted positive health effects, this diet impaired the muscle fatty acid profile, suggesting adverse impacts on human health.

This study aimed to verify the effect of different feeding and stocking conditions during 14 days on the gene expression of several hormones and enzymes related to the stress cascade and metabolic parameters in silver catfish Rhamdia quelen under the following experimental conditions: 1) fed at low stocking density (2.5 kg m -3 , LSD-F); 2) fed at high stocking density (32 kg m -3 , HSD-F); 3) food-deprived at LSD (LSD-FD); and 4) food-deprived at HSD (HSD-FD). Fish from LSD-F and HSD-F groups were fed daily (1 % of their body mass), while fish from food-deprived groups (LSD-FD and HSD-FD) were not fed during the experimental time. Plasma metabolic parameters (glucose, lactate, triglycerides, and proteins) and hepatosomatic index (HSI) were evaluated. In addition, mRNA expression of genes related to the stress axis (crh, pomca, pomcb, nr3c2, star, hsd11b2 and hsd20b), heat shock protein family (hsp90 and hspa12a), sodium-dependent noradrenaline transporter (slc6a2), and growth axis (gh and igf1) were also assessed. Specific growth rate and HSI decreased in food-deprived fish regardless of stocking density. The HSD-FD group showed weight loss compared to the HSD-F, LSD-F, and LSD-FD groups. Plasma glucose and triglycerides were reduced in food-deprived groups, while lactate and protein levels did not change. The expression of key players of the stress response (crh, pomca, pomcb, hsd11b2, nr3c2, and hsp90b) and growth (gh and igf1) pathways were differently regulated depending on the experimental condition, whereas no statistical difference between treatments was found for hsd20b, scl6a2, hspa12a, and star mRNAs expression. This study suggests that LSD acts as a stressor affecting negatively the physiological status of fed fish, as demonstrated by the reduction in growth rates, altered metabolic orchestration, and a higher crh mRNA expression. In addition, food deprivation also increased mRNA expression of other assessed genes (nr3c2, hsp90b, pomca, and pomcb) in fish from the HSD group, indicating higher responsiveness to stress in this stocking density when combined with food deprivation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This study aimed to evaluate different concentrations of the essential oil of Hesperozygis ringens (EOHR) and its effects on anesthesia and transport of Oreochromis niloticus. Experiment I evaluated the concentrations of 0, 150, 300, 450, and 600 µL L -1 EOHR for times of induction and recovery from anesthesia and ventilatory frequency (VF) of O. niloticus (26 g), with 10 repetitions each in a completely randomized design. Based on the results of Experiment I, Experiment II submitted fish (25 g) to three treatments-control (clean water), ethanol (5 mL ethyl alcohol), and 600 µL L -1 EOHR-and then handling for biometry. Blood was collected 1 and 24 h after exposure and handling to analyze hematological and biochemical parameters in a completely randomized design in a factorial arrangement (3 × 2). Experiment III submitted fish (35 g) to simulated transport (4.5 h) with 0, 10, or 20 µL L -1 EOHR and determined the effects on blood variables. Concentrations of 450 and 600 µL L -1 EOHR provoked deep anesthesia in juvenile O. niloticus and provided induction and recovery times within the limits considered ideal for fish. However, this essential oil was not able to attenuate the effects of stress caused by biometric handling. EOHR was able to attenuate the effects of stress from simulated transport, with 10 µL L -1 EOHR being responsible for causing a decrease in protein, triglycerides, and cholesterol values immediately after transport of O. niloticus., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The physiological and behavioral responses of Pomacea canaliculata exposed to different pH values (4, 5, 6, 7, 8, 9, and 10) were evaluated. Survival, behavior (avoidance), metabolites (mantle), net ion fluxes (Na + and K + ), and ATPase activity (gills) were the parameters analyzed. The final survival rates were 100% (pH 4-9) and 90% (pH 10), and the groups did not differ significantly. Avoidance behavior was not identified in animals exposed to an extreme pH compared to pH 7. The main changes observed in the metabolites were in those exposed to an alkaline pH. Glucose (pH 9) and total protein (pH 9 and 10) levels increased, and lactate decreased (pH 9 and 10) compared to a neutral pH. There was an increase in Na + efflux at pH 4, 5, and 8 and an influx at pH 9 and 10. Extreme pH values (4 and 10) also caused an increase in K + efflux. At pH values outside the neutrality range (pH 7), there was a significant decrease in the activities of Na + /K + -ATPase (4, 5, 6, 9, and 10) and H + -ATPase (pH 4, 5, 9, and 10). Variations in environmental pH did not cause statistically significant mortality or avoidance behavior in P. canaliculata at the analyzed times. However, due to changes in energy metabolism (glucose and lactate, mainly) and ionoregulation, these can be considered sensitive biomarkers of stress in this species., (Copyright © 2022. Published by Elsevier Inc.)

The aim of this study was to evaluate the toxicity of tucumã oil nanocapsules from the Amazon region in silver catfish, Rhamdia quelen . Fish were exposed to water treated with different concentrations of tucumã nanocapsules, white, solubilized oil and surfactant vehicles. After three days of exposure, fish were euthanized and liver, gills and brain removed for analysis of the dichlorofluorescein, nitric oxide and PicoGreen® assays. Plasma was collected for assay of hepatic transaminases. The nanocapsules had a diameter of 221 ± 1.27 nm, confirmed by atomic force microscopy. The oil nanocapsules were not toxic to this species of fish, but white nanocapsules and surfactant increased the levels of reactive oxygen species. Thus, nanocapsules are promising for the transport of tucumã oil. In view of the anti-inflammatory properties of this oil, it is possible to envisage its application in skin diseases for example, since they present essentially inflammatory conditions.HighlightsThe most abundant carotenoid in tucumã oil was all - trans -beta-carotene.Nanocapsules are good carriers for tucumã oil.Tucumã oil nanocapsules does nothas toxicity effect in catfish.

Objective: To verify the efficacy of citral in inducing sedation and anesthesia in silver catfish (Rhamdia quelen) and grass carp (Ctenopharyngodon idella) and to assess the safety of essential oil (EO) of Aloysia citriodora and citral in inducing and maintaining anesthesia in silver catfish., Study Design: Clinical study, randomized, parallel, multi-arm with control group in target species., Animals: A total of 96 juvenile and 72 adult silver catfish and 80 juvenile grass carp were used., Methods: Silver catfish and grass carp were exposed to different concentrations of citral, 15-675 and 15-600 μL L -1 , respectively, during the maximum period of 30 minutes to verify sedation and anesthesia induction and recovery times. In addition, for anesthetic induction, silver catfish were exposed to the EO of A. citriodora and citral at 225 μL L -1 for 3.5 minutes. Then, fish were transferred to an anesthesia maintenance solution at 50 μL L -1 for 10 minutes to assess hematologic and biochemical variables at 60 minutes, 2 and 6 days after treatment., Results: Citral only induced sedation from 15, 25 and 40 μLL -1 in both species. Anesthesia without mortality was induced in silver catfish at 50-600 μL L -1 and grass carp at 75-450 μL L -1 . At 675 and 600 μL L -1 , mortality was recorded in silver catfish and grass carp, respectively. The EO of A. citriodora and citral were safe in inducing and maintaining anesthesia in silver catfish, with mean corpuscular hemoglobin concentration being the only variable that varied in relation to time and treatments., Conclusions and Clinical Relevance: Citral was effective in inducing sedation and anesthesia in both species. In addition, A. citriodora EO and citral were safe in inducing and maintaining anesthesia in silver catfish. Both agents are promising substances for the development of new drugs for fish., (Copyright © 2021. Published by Elsevier Ltd.)

This study assessed the potential of eugenol and the essential oil of Lippia alba (EOLA) in providing suitable anesthetic induction and recovery times, and their consequent effects on the blood and respiratory physiology, as well as the gill architecture of an Amazonian freshwater stingray, Potamotrygon wallacei, shortly after reaching the recovery and 48 h later. Juveniles of P. wallacei were exposed to increasing concentrations of eugenol (75, 100, 125, and 150 µL L -1 ) and EOLA (150, 175, 200, and 225 µL L -1 ) in an immersion bath. Anesthetic induction was found to be faster with the use of eugenol compared to EOLA. On the other hand, the stingrays anesthetized with eugenol displayed a longer recovery time than those exposed to EOLA. The highest concentrations of eugenol caused moderate to severe histological changes in the gills. No significant changes were found for hematocrit and plasma energy metabolites in the stingrays anesthetized with all concentrations of both eugenol and EOLA shortly after reaching the recovery from the ansthesia, when compared to those recovered after 48 h. Investigations regarding the potential use of these natural anesthetics are unprecedented for freshwater stingray species, and 200 μL L -1 EOLA is recommended as the most suitable anesthetic for use in juveniles of P. wallacei., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The aim of the current study is to investigate whether silver catfish (Rhamdia quelen) individuals exposed to commercial formulation of the chlorantraniliprole insecticide used in rice crops present changes in biochemical parameters. Fifty-four (54) silver catfish individuals were distributed in six units per tank (n = 6/repetition; triplicate/treatment) and subjected to the following treatments: T1-control, without insecticide; T2 (0.02 µg/L of insecticide) and T3 (0.20 µg/L of insecticide). Exposure time lasted 24 or 96 h, and it was followed by 96 h recovery in pesticide-free water. Results have indicated biochemical changes in cortisol, glucose, lactate and plasma protein levels, as well as few ionic changes in animals' gills during the exposure and recovery periods. Chlorantraniliprole incidence in water resulted in some biochemical changes in silver catfish specimens' plasma and gills throughout the acute exposure protocol (sub-lethal dose). Thus, chlorantraniliprole insecticide has caused osmoregulatory and/or biochemical imbalance in the investigated species under the herein adopted laboratory conditions; these changes did not get back to normal levels even after specimens were left to recover for 96 h in clean water., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The objective of this study was to evaluate the possible relaxing effect of essential oils (EOs) (Aloysia triphylla and Lippia alba) and phytochemicals (citral and linalool) in the gastropod Pomacea canaliculata. Animals were exposed to compounds at the concentrations range of 25-750 µL L-1. Magnesium chloride (MgCl2, 10-50 g L-1) and control group (ethanol 6.75 mL L-1, highest concentration used for treatment dilution) were also tested. The EOs, citral and MgCl2 had no relaxing effect at the concentrations range tested, and citral caused aversive behavior (closure of the operculum) from 90 μL L-1. Exposure to linalool at 25, 50, 100, 200 and 400 µL L-1 relaxed 28, 76, 88, 96 and 100% of the animals, respectively. The concentrations of 25, 50 and 400 µL L-1 differed statistically from each other, while 100 and 200 µL L-1 were equal to 50 and 400 µL L-1. All animals recovered up to 40 min, except at of 400 µL L-1. Linalool is effective for relaxing P. canaliculata and can be useful in management techniques that require relaxation. However, further studies are needed to certify whether linalool is appropriate for maintaining animal welfare in invasive procedures that require total insensitivity.

Lactococcosis, particularly that caused by Lactococcus garvieae , is a major re-emerging bacterial disease seriously affecting the sustainability of aquaculture industry. Medicinal herbs and plants do not have very much in vitro antagonism and in vivo disease resistance towards lactococcosis agents in aquaculture. Most in vitro studies with herbal extractives were performed against L. garvieae with no strong antibacterial activity, but essential oils, especially those that contain thymol or carvacrol, are more effective. The differences exhibited by the bacteriostatic and bactericidal functions for a specific extractive in different studies could be due to different bacterial strains or parts of chemotypes of the same plant. Despite essential oils being shown to have the best anti- L. garvieae activity in in vitro assays, the in vivo bioassays required further study. The extracts tested under in vivo conditions presented moderate efficacy, causing a decrease in mortality in infected animals, probably because they improved immune parameters before challenging tests. This review addressed the efficacy of medicinal herbs to lactococcosis and discussed the presented gaps.

The aim of this review was to summarize the current knowledge regarding the effects of aeromonosis on fish oxidative status. The bibliographic survey was carried out on the research platforms: Scopus and Science Direct. The keywords 'Aeromonas', 'fish' and 'oxidative status' (or 'oxidative stress', 'oxidative damage' and similar terms) were used. Scientific papers and short communications were considered. Studies involving fish aeromonosis and enzymatic or non-enzymatic markers of oxidative status were selected. The results of antioxidant enzymes activities/expressions after infection lack consistency, suggesting that these findings should be interpreted with caution. Most of the analysed studies pointed to an increase in reactive oxygen species, malondialdehyde and protein carbonylation levels, indicating possible oxidative damage caused by the infection. Thus, these three biomarkers are excellent indicators of oxidative stress during infection. Regarding respiratory burst activity, several studies have indicated increased activity, but other studies have indicated unchanged activity after infection. Nitric oxide levels also increased after infection in most studies. Therefore, it is suggested that the fish's immune system tries to fight a bacterial infection by releasing reactive oxygen and nitrogen species., (© 2020 The Society for Applied Microbiology.)

An increase in water temperature in the Amazon River has elicited concerns about commercially important fish species associated with food security, such as matrinxã (Brycon amazonicus). Studies have demonstrated the positive effects of diets supplemented with plant-based products that combat heat stress-induced oxidative damage. The aim of this study was to determine whether dietary supplementation with nerolidol prevents or reduces muscle oxidative damage and impairment of the fillet fatty acid profile of matrinxã exposed to heat stress. Plasma and muscle reactive oxygen species (ROS) and lipid peroxidation (LPO) levels were significantly higher in fish exposed to heat stress compared to fish not exposed to heat stress, while plasma superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity was significantly lower. The total content of saturated fatty acids (SFA) in fillets was significantly higher in fish exposed to heat stress compared to fish not exposed to heat stress, while he total content of polyunsaturated fatty acids (PUFA) was significantly lower. Nerolidol prevented the increase of muscle LPO and plasma ROS and LPO levels in fish exposed to heat stress, and partially prevented the increase in muscle ROS levels. Diets containing nerolidol prevented the inhibition of muscle GPx activity in fish exposed to heat stress, and partially prevented the decrease of plasma GPx activity. The nerolidol-supplemented diet prevented the increase of fillet SFA in fish exposed to heat stress, while partially preventing the decrease of PUFA. We conclude that acute heat stress at 34 °C for 72 h causes plasma and muscular oxidative damage, and that homeoviscous adaptation to maintain membrane fluidity can represent a negative impact for fish consumers. A nerolidol diet can be considered a strategy to prevent heat stress-induced oxidative damage and impairment of muscle fatty acid profiles., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Evidence suggests the involvement of purinergic signaling, a mechanism mediated by extracellular nucleotides and nucleosides, with the impairment of immune and inflammatory responses in silver catfish (Rhamdia quelen) exposed to trichlorfon. Plant-derived substances have been considered potent anti-inflammatory agents due to effects on the purinergic system, such as the use of the flavonoid rutin. The aim of this study was to determine whether a diet containing rutin is able to prevent or reduce trichlorfon-induced impairment of immune responses through alteration of the purinergic pathway. Spleen adenosine triphosphate (ATP) levels were significantly higher in silver catfish exposed to 11 mg/L trichlorfon for 48 h compared to the control group, while adenosine (Ado) levels were significantly lower. Spleen ectonucleoside triphosphate diphosphohydrolase (NTPDase) activity was significantly lower in silver catfish exposed to trichlorfon compared to control group, while adenosine deaminase activity was significantly higher. Spleen metabolites of nitric oxide, interleukin-1, and IL-6 were significantly higher in silver catfish exposed to trichlorfon compared to control group. Diet with 3 mg rutin/kg diet was able to prevent all the alterations elicited by trichlorfon, except restoring spleen ATP levels. The purinergic exposure signaling is involved in impairment of immune and inflammatory responses in fish exposed to trichlorfon due to reduction in ATP hydrolyses and by an increase in Ado deamination, leading to release of pro-inflammatory mediators. Use of rutin-added diet exerted an essential role in protecting the silver catfish spleen from trichlorfon-induced impairment on immune and inflammatory responses, preventing all alterations on splenic purinergic signaling., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Amazonian aquatic ecosystem undergoes seasonal variations and daily changes that directly affect the availability of oxygen. During the day the levels of oxygen can reach supersaturation, and at night can drop to zero. In this way, aquatic organisms are exposed daily to physiological challenges regarding the availability of oxygen. The present study revealed significant differences in the physiology and performance of two cichlids: Geophagus proximus (black water cichlid - from Negro River) and Chaetobranchopsis orbicularis (white water cichlid - from Amazon River), exposed to hypoxia. The white water cichlid showed lower value (1.99 ± 0.79 pKa) of critical pressure of oxygen (Pcrit) and a longer time (68.00 ± 14.11 min) for total loss of balance (LOE); however, this species showed 50% mortality during exposure to hypoxia, while the black water cichlid did not show mortality. Both cichlids presented a decrease in O 2 consumption rate (OCR) during hypoxia.. In this sense, it was observed that the black water cichlid presented several physiological strategies during hypoxia, such as, a significant increase in plasma cortisol levels, nucleoside triphosphate diphosphohydrolase activity (for adenosine diphosphate (ADP) as a substrate) in the gills, and the activity of adenosine deaminase (ADA) in gills and liver, in addition to a significant increase in the activity of complexes (II-III) in the transporter chain of electrons in both analyzed tissues and succinate dehydrogenase activity of gills' mitochondria. On the other hand, the only physiological change observed in the white water cichlid was a significant reduction in the activity of complexes II-III in gills and liver. Based on our findings, we can hypothesize that the white water cichlid specie has less tolerant to hypoxia when compared to the black water cichlid., (Copyright © 2021. Published by Elsevier Inc.)

This study points to evaluate the effects of pre-treatment with standardized dry extract of Curcuma longa (Motore™) added to the diet (0; 250; 500; and 750 mg/kg) on oxidative stress parameters, longevity, and therapeutic success in Rhamdia quelen experimentally infected with Aeromonas hydrophila (MF 372510). After treatment, the liver and kidney were collected to determine non-enzymatic oxidative parameters such as the formation of thiobarbituric acid reactive substances (TBARS), non-protein thiols (NPSH), and quantification of reactive oxygen species (ROS) levels. Also, two enzymatic antioxidant parameters were evaluated: superoxide dismutase (SOD) and catalase (CAT) activities. The results showed an increase of ROS and TBARS levels, a depletion in NPSH, and a decrease of SOD and CAT activities in infected fish compared to control. The highest Motore™ dose minimized the deleterious effect of A. hydrophila infection improving longevity, oxidative status, and survival rate. The addition of 750 mg Motore™/kg feed is recommended for silver catfish in fish farming. Serious economic losses in Rhamdia quelen culture caused by Aeromonas hydrophila infections can be prevented by the addition of Motore™ to the diet., (Copyright © 2021. Published by Elsevier Ltd.)

This study aimed to evaluate the essential oil of Ocimum gratissimum L. (EOOG) for anesthesia and in the transport of Oreochromis niloticus. Experiment I determined the time of anesthesia induction and recovery during anesthesia of O. niloticus exposed to different concentrations of EOOG (0, 30, 90, 150, and 300 mg L -1 ). Based on data from Experiment I, Experiment II evaluated the effect of 0, 30, and 90 mg L -1 EOOG on blood parameters and oxidative stress immediately after anesthesia induction and 1 h after recovery. Experiment III evaluated the effect of 0, 5, and 10 mg L -1 EOOG on blood variables immediately after 4.5 h of transport of juveniles. Concentrations between 90 and 150 mg L -1 EOOG were efficient for anesthesia and recovery. The use of 90 mg L -1 of EOOG prevented an increase in plasma glucose. Other changes in blood parameters and oxidative stress are discussed. The use of 10 mg L -1 EOOG in transport increased plasma glucose and decreased hematocrit values immediately after transport. It is concluded that the use of 90 and 150 mg L -1 EOOG causes anesthesia and recovery in O. niloticus within the time intervals considered ideal. The use of 90 mg L -1 EOOG favored stable plasma glucose soon after anesthesia induction and 1 h after recovery, but caused changes in the antioxidant defense system by increasing hepatic and kidney ROS. The transport of 12 g O. niloticus for 4.5 h can be performed with concentration of 5 mg L -1 of EOOG.

Trichlorfon is an organophosphate pesticide used extensively for controlling ectoparasites in aquaculture. Studies have found that trichlorfon caused environmental pollution and severe neurotoxic effects in several freshwater species. Feed additives such as flavonoids may reduce or prevent pesticide-induced toxicity in fish. The aim of the present study was to determine whether acute exposure to trichlorfon impairs behavior and causes oxidative damage in brains of silver catfish (Rhamdia quelen). We also sought to determine whether rutin would be capable of preventing or reducing these effects. Silver catfish were divided into four groups: groups A and C received basal feed, while groups B and D received feed containing 3 mg rutin/kg diet for 21 days. After 21 days, groups C and D were exposed for 48 h to a nominal concentration of 11 mg trichlorfon/L water. Fish exposed to trichlorfon showed significantly longer distances travelled and swimming performances than did unexposed fish. Cerebral levels of reactive oxygen species and lipid peroxidation were significantly higher in fish exposed to trichlorfon than in unexposed fish, while cerebral superoxide dismutase, catalase, glutathione peroxidase, and acetylcholinesterase (AChE) activities were significantly lower. Taken together, our findings suggest that dietary supplementation rutin completely prevented all alterations elicited by trichlorfon, except for cerebral AChE activity; the latter remained significantly lower compared to the unexposed group. In summary, rutin prevents trichlorfon-induced neurotoxicity in silver catfish., (Copyright © 2020 Elsevier Inc. All rights reserved.)

It is unknown whether the flavonoid rutin can protect the silver catfish liver in response to exposure to a known stressor, such as the prophylactic usage of the antimicrobial agent oxytetracycline. Thus, the current study aimed to assess the effect of rutin incorporation into the silver catfish diet formulation on oxytetracycline-induced liver oxidative stress and apoptosis. Fish were split into four groups as follows: control, rutin (1.5 g kg diet -1 ), oxytetracycline (0.1 g kg diet -1 ) and rutin+oxytetracycline (1.5 g kg diet -1 and 0.1 g kg diet -1 , respectively). After two weeks of feeding with the different diets (standard, rutin-, oxytetracycline and rutin+oxytetracycline-added diets), fish were euthanized to collect the liver. Although the rutin-added diet was unable to recover glutathione peroxidase activity, ascorbic acid and reduced glutathione (GSH) levels, which were depleted due to oxytetracycline consumption, it markedly diminished the oxidized glutathione (GSSG) content, thus decreasing the GSSG to GSH ratio, an important index of oxidative stress. It also increased glutathione reductase and markedly augmented glucose-6-phosphate dehydrogenase activities, which were declined after oxytetracycline ingestion. Furthermore, the rutin-added diet reestablished superoxide dismutase and catalase activities and reduced lipid peroxidation, nitric oxide and superoxide anion levels as well, all changes resulting from oxytetracycline consumption. Finally, it also prevented oxytetracycline-induced apoptosis through increasing heat shock protein 70 and markedly decreasing high mobility group box 1 and, consequently, reducing cleaved caspase-3 protein levels. Therefore, in conclusion, the incorporation of this flavonoid to the silver catfish diet protected the liver against oxytetracycline-induced liver oxidative stress and apoptosis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rapid industrialization results in the production of large quantities of waste that are commonly discharged into water bodies, leading to the damage of the aquatic ecosystem and freshwater organisms. Copper (Cu) can induce oxidative damage in fish muscle, the main fish portion that is consumed by humans. However, the responses of the Amazon fish Cichlasoma amazonarum and its capacity to withstand acute Cu concentrations found in Amazon water around mines remain unknown. Thus, the aim of this study was to evaluate whether exposure to Cu causes muscle oxidative stress and/or oxidative damage and impairs the fillet fatty acid profile of C. amazonarum acutely exposed to Cu found in Amazon waters around mines. Muscle reactive oxygen species and protein carbonylation levels were significantly higher in fish exposed to 1500 μg/L Cu compared with the control group, while muscle lipid peroxidation levels were significantly higher in fish exposed to 500, 750, and 1500 μg/L Cu compared with control group. Muscle antioxidant capacity against peroxyl radical's levels and glutathione peroxidase activity were significantly lower in fish exposed to 1500 μg/L Cu compared with the control group, while muscle superoxide dismutase activity was significantly lower in fish exposed to 750 and 1500 μg/L Cu compared with control group. The total content of saturated fatty acids was significantly higher in fish exposed to 1500 μg/L Cu compared with the control group, while the total content of monounsaturated fatty acids and sum of n3 fatty acids were significantly lower in fish exposed to 1500 μg/L Cu compared with control group. No significant difference was observed regarding muscle catalase, glutathione S-transferase, and glutathione reductase activities. Based on these lines of evidence, the results of this comprehensive study agree with the initial hypothesis that the exposure to Cu found in Amazon water around mines induces oxidative damage and inhibits enzymatic and non-enzymatic antioxidant response in the muscle of C. amazonarum exposed to high Cu levels. Moreover, the impairment of the fillet fatty acid profile appears to be mediated by oxidative damage, representing a negative impact on fish health.

This study aimed to verify whether dietary quercetin protects against the detrimental effects induced by oxytetracycline (OTC) administration in silver catfish (Rhamdia quelen). Fish were divided into different experimental groups that received OTC and/or quercetin, either during 14 or 21 days. To determine the endocrine system stress response, we have measured the brain mRNA expression levels of corticotropin-releasing hormone (crh), proopiomelanocortins (pomca and pomcb) and some of the pituitary hormones (growth hormone [gh], somatolactin [sl], and prolactin [prl]). We have also quantified the levels of cortisol as well as some metabolites (glucose, glycogen, lactate, and triglycerides) in the plasma. Moreover, the enzymatic activity of hexokinase, phosphorylase (active GPase), fructose-biphosphatase (FBP), glycerol-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate dehydrogenase (GDH) and gill Na + /K + -ATPase were measured. The results demonstrated that OTC activates the silver catfish stress response by increasing the plasma cortisol and decreasing the glucose levels at 14 and 21 days. Additionally, OTC also altered the fish hepatic metabolic status as demonstrated by an increase in triglycerides levels and the enzymatic activity of both FBP and GDH after 14 days. OTC also stimulated Na + /K + -ATPase activity in the gill after 14 days and altered the hypophyseal expression of gh (at 14 and 21 days) and prl (at 14 days). The co-treatment with 1.5 g of quercetin could prevent most of the alterations caused by OTC, strongly suggesting quercetin as a beneficial compound when added to the fish diet., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Inc.)

Trichlorfon is an organophosphate insecticide that is widely used on fish farms to control parasitic infections. It has been detected in freshwater ecosystems as well as in fishery products. There is a growing body of evidence to suggest that certain feed additives may reduce or prevent pesticide-induced toxicity in fish. The aim of the present study was to determine whether acute exposure to trichlorfon would alter bioenergetic homeostasis and alter fatty acid profiles in muscles of silver catfish (Rhamdia quelen). We also sought to determine whether rutin prevents or reduces these effects. Cytosolic and mitochondrial creatine kinase (CK) and activities of complexes II-III and IV in muscle were significantly inhibited by exposure to 11 mg/L trichlorfon for 48 h compared to effects in the unexposed group. Total content of polyunsaturated fatty acids (omega-3 and omega-6) were significantly lower in muscle of silver catfish exposed to 11 mg/L trichlorfon for 48 h than in the unexposed group. Addition of 3 mg rutin/kg feed increased CK activity and prevented inhibition of complex IV activity, as well as preventing all alterations of muscle fatty acid profiles elicited by exposure to trichlorfon. No significant differences were observed between groups with respect to muscle adenylate kinase or pyruvate kinase activities, as well as total content of saturated and monounsaturated fatty acids. Our findings suggest that exposure (48 h) to 11 mg trichlorfon/L water inhibits cytosolic and mitochondrial CK activity in muscle. Trichlorfon also affects activities of complexes II-III and IV in respiratory chain, with important consequences for adenosine triphosphate production. The pesticide alters fatty acid profiles in the fish and endangers human consumers of the product. The most important finding of the present study is that inclusion of rutin improves bioenergetic homeostasis and muscle fatty acid profiles, suggesting that it reduces trichlorfon-induced muscle damage., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The practice of replacing costly animal-derived proteins with more economical plant proteins has augmented the risk of mycotoxin contamination in fish feeds, including contamination with ochratoxin A (OTA). OTA is a secondary metabolite produced by molds commonly found in fish feeds that causes impairment of performance in several fish species and some hepatic biochemical alterations. However, the pathways involved in hepatic damage remain unknown and are limited to histopathological alterations. Purinergic signaling is a homeostatic system that continuously monitors the internal environment to detect injury primarily by two intercellular messengers: adenosine triphosphate (ATP) and adenosine (Ado). The objective of this study was to determine whether OTA-contaminated feed induces the release of nucleotides in the extracellular milieu, as well as whether ectoenzymes modulate ATP pro-inflammatory effects in liver of tambaqui (Colossoma macropomum). Final mean weight, weight gain (WG), and liver weight were significantly lower in tambaqui fed feeds containing 1.6 and 2.4 mg OTA/kg feed than in the control group. Liver ATP and Ado levels were significantly higher in tambaqui fed feeds containing 1.6 and 2.4 mg OTA/kg feed compared with control, while no significant difference was observed regarding adenosine diphosphate and adenosine monophosphate levels. Hepatic triphosphate diphosphohydrolase (NTPDase) activity (for ATP) was significantly greater in tambaqui fed feeds containing 1.6 and 2.4 mg OTA/kg feed compared with control, while adenosine deaminase (ADA) activity was lower. No significant difference was observed with respect to hepatic NTPDase activity (for ADP) or for 5'-nucleotidase activity. Finally, levels of liver metabolites of nitric oxide were significantly higher in tambaqui fed feeds containing 1.6 and 2.4 mg OTA/kg feed than in the control group. Based on these data, exposure to 1.6 and 2.4 mg OTA/kg feed impaired tambaqui growth performance associated with final mean weight and WG. Levels of two important intercellular messengers, ATP and Ado, increased in the extracellular space as a consequence of hepatic damage, exerting opposite immune responses. Finally, liver NTPDase and ADA activities were altered to modulate ATP and Ado levels, respectively, exerting anti-inflammatory effects to counteract OTA-induced hepatic injury.

The toxic effects of copper (Cu) are linked to dysfunction of metabolism and depletion of adenosine triphosphate (ATP). Nevertheless, the effects related to phosphoryl transfer network, a network of enzymes to precise coupling of the ATP-production and ATP-consuming process for maintenance of bioenergetic, remain unknown. Therefore, the aim of this study was to determine whether the phosphoryl transfer network could be one pathway involved in the bioenergetic imbalance of Cichlasoma amazonarum exposed for 96 h to environmentally relevant concentrations of Cu found in Amazonia water around mines. Branchial mitochondrial creatine kinase (CK) activity was significantly lower in fish exposed to 1500 μg/L Cu than in the control group, while branchial cytosolic CK activity was significantly greater. Branchial (exposed to 750 and 1500 μg/L Cu) and hepatic (exposed to 1500 μg/L Cu) pyruvate kinase (PK) activity was significantly lower in fish exposed to Cu than in the control group. Branchial and hepatic ATP levels were significantly lower in fish exposed to 1500 μg/L than in the control group. Branchial reactive oxygen species (ROS) and lipid peroxidation (LPO) levels were significantly higher in fish exposed to 750 and 1500 μg/L Cu compared to control. Hepatic ROS and LPO levels were significantly higher in fish exposed to 1500 μg/L than in the control group. Branchial and hepatic Cu levels were significantly higher in fish exposed to 1500 μg/L compared to other groups. Exposure to 750 and 1500 μg/L Cu impairs bioenergetics homeostasis, which appears to be mediated by ROS overproduction and lipid peroxidation., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Amazon fish are vulnerable to climate change. Current temperature increases in Amazonian rivers are likely to continue in the coming years. Elevated temperatures impair homeostasis and subject fish to oxidative stress; nevertheless, the effects of elevated temperature on plasma and muscle oxidative stress as well as fillet fatty acid composition in matrinxã (Brycon amazonicus) remain unknown. Therefore, the aims of the present study were to determine whether antioxidant system is able to withstand acute thermal stress to avoid plasma and muscle oxidative damage and to determine the manner in which matrinxã adjust their muscle fatty acid metabolism in a response to acute heat stress. We exposed juvenile matrinxã to four temperatures (28 °C as control and 30, 32, and 34 °C) for 72 h and observed the effects on plasma and muscle oxidant/antioxidant status and on fatty acid composition. Plasma reactive oxygen species (ROS) and lipid peroxidation (LPO) levels were significantly higher in matrinxã exposed to 34 °C compared with the control group, while they were significantly higher in the muscle of matrinxã exposed to 32 °C and 34 °C compared with the control group. Superoxide dismutase (SOD) activity and total antioxidant capacity (TAC) levels were significantly lower in the plasma and muscle of matrinxã exposed to 34 °C compared with the control group. Plasma glutathione peroxidase (GPx) activity was significantly lower in matrinxã exposed to 32 and 34 °C compared with the control group, while it was significantly lower in the muscle of matrinxã exposed to 34 °C. The total content of saturated fatty acid (SFA) was significantly higher in the fillet of matrinxã exposed to 34 °C compared with the control group, while the total content of polyunsaturated fatty acid (PUFA) was significantly lower. Based on this evidence, we conclude that acute heat stress at 32 °C and 34 °C causes plasma and muscular oxidative stress, and the enzymatic and non-enzymatic antioxidant system is unable to prevent oxidative damage. Moreover, increased levels of total SFA and decreased levels of PUFA occur in an attempt to withstand heat stress and maintain membrane fluidity; nevertheless, these responses can represent negative impacts for consumers.

Plant-based ingredients are successfully replacing fishmeal in fish feeds. However, this practice increases the risk of feed contamination by mycotoxins that reduce production and heath associated with oxidative damage. The aim of this study was to determine whether feed contaminated with environmentally relevant concentrations of ochratoxin A (OTA) causes muscle oxidative damage in tambaqui (Colossoma macropomum), and to determine whether this feed impairs fatty acid profiles in fish meat. Final mean weight, weight gain, daily weight gain, feed efficiency, and specific growth rate were significantly lower in fish fed 1.6 and 2.4 mg OTA/kg compared to those fed basal diet, while feed conversion was significantly higher in these same groups compared to the basal group. Levels of reactive oxygen species and lipid peroxidation in muscle were significantly higher in fish fed 1.6 and 2.4 mg OTA/kg compared to those in the basal group. Likewise, muscle superoxide dismutase and glutathione peroxidase activities were significantly higher in fish fed 1.6 and 2.4 mg OTA/kg then in those fed basal feed. The total content of saturated fatty acids was significantly higher, and total content of polyunsaturated fatty acids was significantly lower in fish fed 2.4 mg OTA/kg compared to those fed basal feed. Taken together, the data suggest that OTA-contaminated feed induces oxidative damage and disturbs enzymatic and non-enzymatic antioxidant responses in tambaqui meat. The perturbations of fatty acid profiles in fish meat appears to mediated by oxidative damage, representing negative impact on fish health and presenting concerns for consumers of fish., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) is a mediator and biomarker of pro- and anti-inflammatory processes. Excessive levels of NO for long periods have been associated with inflammation and tissue damage. The metabolism and synthesis of NO is usually measured indirectly, as metabolites and enzymes involved in reactions, often as the nitrite/nitrate (NOx) level. The aim of the present study was to measure the NOx levels in vital organs of juvenile silver catfish (Rhamdia quelen) exposed to various levels of eprinomectin in the water. The fish were exposed for 24 and 48 h to start concentration (0 h) of eprinomectin in water (0.0, 1.12, 1.80, and 3.97 μg/L). The eprinomectin concentrations in water were lower at 24 h (0.0, 0.85, 1.14, and 1.15 μg/L) and 48 h (0.0, 0.39, 0.69, and 1.28 μg/L), due to the process of eprinomectin metabolization. Subsequently, the fish were left for 48 h of recovery in eprinomectin-free water. NO levels were measured indirectly, as NOx levels in brain, liver, and gill tissue. Within 24 h of exposure, there was no significant increase in NOx levels in the organs evaluated at any of the concentrations tested. However, increases in NOx levels did occur at 48 h of exposure in all organs, particularly at the two highest concentrations of eprinomectin (1.80 and 3.97 μg/L). The transfer of fish to eprinomectin-free water did not result in reversal of NOx levels after 48 h of recovery, especially in fish that had been exposed to the two highest concentrations in the brain and liver tissues, and for the highest concentration in the gills. We conclude that silver catfish exposed to eprinomectin for up to 48 h present possible cerebral, hepatic, and branchial inflammatory process associated with increased tissue NOx levels, and that recovery for 48 h in water without antiparasitic is insufficient for the fish to recover from the poisoning.

Stress responses in teleosts include the release of hormones into the bloodstream. Their effects depend on the species and on the environmental conditions. The Amazon basin collects waters of diverse chemical composition, and some fish are able to inhabit several of them. However, the effects of these waters on the stress axis are still unknown. Here we show how acute air-exposure differently affects stress biomarkers in tambaqui (Colossoma macropomum), a tropical model species, when acclimated to two Amazonian waters (Rio Negro -RN- water rich in humic acids and poor in ions, and groundwater -IG- with no humic acids and higher concentration of ions). This study described primary and secondary stress responses after air exposure including plasma cortisol, energy metabolites, pH and ions, skin mucus energy metabolites, as well as gills and kidney Na + /K + -ATPase and Na + /H + -exchanger (NHE) activities. Several differences were described in these stress biomarkers due to the acclimation water. The most remarkable ones include increased mucus glucose only in RN-fish, and mucus lactate only in IG-fish after air exposure. Moreover, an inverse relationship between plasma cortisol and Na + concentrations as well as a direct relationship between plasma ammonia and branchial NHE activity were observed only in RN-fish. Our results demonstrate how important is to study stress responses in fish acclimated to different environments, as physiological differences can be magnified during episodes of high energy expenditure. In addition to having a direct application in aquaculture, this study will improve the management of critical ecosystems such as the Amazon., Competing Interests: Declaration of Competing Interest The authors declare no competing or financial interests. The funding sponsors had no role in the design of the study, in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nerolidol is a sesquiterpene found in essential oils of several plant species. It is found commonly in human and animal diets and is approved by the US Food and Drug Administration as a flavoring agent. Nevertheless, recent studies have suggested that nerolidol has potent hepatotoxic effects. Because use of plant-based products in human and animal food has expanded considerably, it is essential to develop approaches such as nanotechnology to avoid or reduce hepatic toxic effects. Therefore, the aim of the study was to determine whether nerolidol dietary supplementation elicited hepatic damage associated with impairment of energy homeostasis, as well as whether supplementation with nerolidol-loaded in nanospheres prevented hepatotoxic effects in Nile tilapia (Oreochromis niloticus). Nile tilapia were divided into five groups (A-E, n = 10 per group) with four replicates each, as follows: group A received basal feed (without supplementation); group B received feed containing 0.5 mL free nerolidol/kg; group C received feed containing 1.0 mL free nerolidol/kg; group D received feed containing 0.5 mL nanospheres nerolidol/kg; and group E received feed containing 1.0 mL nanospheres nerolidol/kg. All groups received experimental feed once a day (10% total biomass) at 2 p.m. for 60 consecutive days. Hepatic liver weight and relative liver weight were significantly lower in fish fed 1.0 mL free nerolidol/kg feed than in fish given basal diet (control group). Hepatic pyruvate kinase (1.0 mL free nerolidol/kg) and adenylate kinase (0.5 and 1.0 mL free nerolidol/kg) activities were significantly lower than in the control group, while hepatic reactive oxygen species and lipid damage levels were significantly higher. Finally, the comet assay revealed significant increases in the frequency of damage and the damage index in fish given 0.5 and 1.0 mL free nerolidol/kg in a dose-dependent manner. Nerolidol-loaded in nanospheres prevented all alterations elicited by free nerolidol. Based on these data, we concluded that dietary supplementation with free nerolidol elicited severe impairment of hepatic bioenergetics homeostasis that appeared to be mediated by excessive ROS production and lipid damage, contributing to a genotoxic effect. Dietary supplementation with nerolidol-loaded in nanospheres did not elicit hepatic damage, and therefore, should be considered as a replacement so as to limit toxicity, permitting its continued use as a dietary supplement.

The trend toward using plant-based ingredients in aquafeeds has raised important concerns for aquaculture owing to the negative impacts of mycotoxins on fish health; with emphasis for contamination by fumonisin B 1 (FB 1 ). The brain is an important target of FB 1 ; however, study of the pathways linked to brain damage is limited to an analysis of histopathological alterations. Reports have demonstrated the protective effects of dietary supplementation with diphenyl diselenide (Ph 2 Se 2 ) in the brains of fish subjected to several environmental insults; nevertheless, its neuroprotective effects in fish fed with diets contaminated with FB 1 remain unknown. Therefore, the aim of this study was to evaluate whether oxidative damage may be a pathway associated with FB 1 -induced neurotoxicity, as well as to evaluate whether dietary supplementation with Ph 2 Se 2 prevents or reduces FB 1 -mediated brain oxidative damage in silver catfish. Brain reactive oxygen species (ROS), lipid peroxidation (LOOH) and protein carbonylation increased on day 30 post-feeding in animals that received FB 1 -contaminated diets compared to the control group, while brain antioxidant capacity against peroxyl radicals (ACAP) levels and catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities were lower. Diphenyl diselenide dietary supplementation avoid increases in brain ROS levels, as well minimizing the augmentation of LOOH levels. Furthermore, Ph 2 Se 2 prevented impairment of brain ACAP levels, as well as GPx and GST activities elicited by FB 1 -contaminated diets. These data suggest that dietary supplementation with 3 mg/kg Ph 2 Se 2 prevented FB 1 -induced brain damage in silver catfish, and this protective effect occurred through avoided of excessive ROS production, as well as via prevention of brain lipid damage. Furthermore, Ph 2 Se 2 exerted its neuroprotective effects via ameliorative effects on the enzymatic and non-enzymatic antioxidant defense systems, and may be an approach to prevent FB 1 -induced brain oxidative stress; however, is not an alternative to prevent the impairment on performance caused by FB 1 ., Competing Interests: Declaration of competing interest None., (Copyright © 2020. Published by Elsevier Inc.)

Rampant and uncontrolled use of antibiotics is a major concern for aquaculture; the practice foments the emergence of resistant strains of Streptococcus agalactiae, among other negative impacts. Constituents of plant essential oils such as nerolidol are being considered as replacements for synthetic drugs to support fish nutrition and health. There is evidence to suggest that nanotechnology may enhance the efficacy of natural bioactive compounds; this is a substantial advance for the development and sustainability of aquaculture. Against the backdrop of this evidence, we aimed determine whether dietary supplementation with free nerolidol and nerolidol-loaded nanospheres would exert bactericidal effects against S. agalactiae, as well as prevent S. agalactiae-induced brain oxidative damage. In Experiment I, we measured the antimicrobial properties of dietary supplementation of nerolidol and nerolidol nanosphere in terms of mortality, longevity and relative percent survival. Fish infected with S. agalactiae fed 0.5 and 1.0 mL nerolidol nanospheres kg/diet demonstrated lower mortality and higher relative percent survival than the control group, while longevity was higher in all infected plus supplementation groups. Experiment II showed significantly lower microbial loads in brains of fish infected with S. agalactiae that were fed 1.0 mL nerolidol nanospheres kg/diet than in the control group. Brain nerolidol levels were significantly higher in uninfected as well as infected fish supplemented with nerolidol nanospheres than in fish supplemented with free nerolidol. Finally, brain reactive oxygen species and lipid peroxidation levels were higher in infected fish supplemented with basal diet compared to uninfected fish and supplemented with basal diet, and the supplementation with 1.0 mL/kg nerolidol nanospheres prevented this augmentation caused by infection. These data suggest that dietary supplementation with nerolidol nanospheres (1.0 mL/kg diet) has potent bactericidal effects in terms of augmentation of fish longevity and survival, and reduction of brain microbial loads. Also, S. agalactiae-induced brain oxidative damage that contributed to disease pathogenesis, and the dietary supplementation with nerolidol nanospheres (1.0 mL/kg diet) prevented this alteration. In summary, nanotechnology is a compelling approach to enhancing the efficacy of nerolidol, giving rise to reduction of S. agalactiae loads in fish brains., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Amazon fish are vulnerable to climate change. Several lines of evidence suggest that the temperature of Amazonian rivers will increase in the coming years. Elevated temperature disturbs homeostasis and subjects fish to physiological stress; however, the effects of temperature on immunity remain poorly understood, particularly those effects involving purinergic signaling. This system fine-tunes the inflammatory and immune responses triggered by stress. Therefore, the aims of this study were to determine whether acute heat stress induces the release of nucleotides into extracellular compartment and to determine whether purinergic enzymes modulate the proinflammatory effects of adenosine triphosphate (ATP) in plasma and spleen of matrinxã (Brycon amazonicus) exposed to acute heat stress. We exposed juvenile matrinxã to four temperature regimes (28 °C as control, 30, 32 and 34 °C) for 72 h and observed the effects on purinergic signaling. Plasma cortisol levels were significantly higher in fish exposed to 34 °C than in the control group, while spleen ATP, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) levels were significantly higher in this group than in controls. Activities of spleen nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase were significantly higher in fish exposed to 34 °C than those of the control group, while spleen interleukin-1 (IL-1) and interleukin-6 (IL-6) levels were higher in this same group than in the control group. No significant differences were observed between the groups regarding plasma parameters. Based on these data, we concluded that acute heat stress at 34 °C caused physiological stress in matrinxã, manifesting as elevated plasma cortisol levels. The most important finding is that purinergic enzymes were modulated, though not efficiently, in response to the excessive release of nucleotides into the extracellular space. In summary, the purinergic signaling pathway may be involved in the impairment of immune and inflammatory responses in matrinxã exposed acutely to 34 °C., (Copyright © 2020 Elsevier Ltd. All rights reserved.)